The present invention relates generally to the manipulation of genetic materials and more particularly to the manufacture of DNA sequences facilitating microbial expression of selected plasmid-borne structural genes.
Transcription is the process whereby genetic information stored in deoxyribonucleic acid polymers (DNA) in the cells is transferred or transcribed into ribonucleic acid polymers called messenger RNAs (mRNAs) which are thereafter translated into proteins. The transcription process is initiated by genetic regulatory regions called promoters which effect interaction of a transcribing enzyme (RNA polymerase) and template, thus controlling the level of transcription activity of adjacent nucleotide sequences. Promoters, which usually preceed protein-coding nucleotide sequences ("structural genes") in a DNA polymer, vary in "strength", depending upon their varied abilities to recognize and bind RNA polymerase in a manner facilitating initiation of mRNA transcription.
Regulation of transcription of certan nucleotide sequences in procaryotic organisms is, in many cases, facilitated by nucleotide sequences called operators. Positioned adjacent to, or interwoven with, the promoter, operator sequences generally function through recognition of modulator proteins. Interaction of the operator DNA sequence with a modulator protein (usually an inducible or repressible enzyme) either activates or represses the associated promoter's transcriptional activities, depending upon the nature of the interaction. It is hypothesized that interaction of the modulator protein with the operator alters the affinity of the promoter sequence for RNA polymerase and/or sterically alters access of RNA polymerase to the promoter sequence.
At least two major factors are therefore responsible for controlling the ability of RNA polymerase to interact with the promoter and thereby effect its transcriptional activity: (1) the inherent strength of the promoter, and (2) the state of regulation which may be imposed upon the promoter by an associated operator sequence interacting with the modulator protein. Sequences which precede a selected gene or series of genes in a functional DNA sequence and which operate to determine whether the transcription and eventual expression of a gene will take place are then collectively referred to as promoter/regulator, promoter/operator, or control DNA sequences.
DNA sequences which follow a gene in a DNA polymer and provide a signal for termination of the transcription into messenger RNA are also significant for expression of a gene and are referred to as terminator sequences.
Recombinant DNA methodologies currently enable genetic transformation of a host microorganism with a DNA (plasmid or viral) vector including an exogenous structural gene coding for a polypeptide whose production in the organism is desired Successful expression of an exogenous gene in a transformed host microorganism depends to a great extent on association of the gene in the transformation vector with a suitable promoter/operator sequence so as to insure high level transcription of the gene into mRNA. It is not often the case that the naturally associated promoter/operator of an exogenous gene will allow for high levels of expression in the new host and thus a gene to be microbially expressed must ordinarily be fitted with a new, host-accommodated transcription regulating DNA sequence prior to insertion.
A variety of bacterial and bacteriophage promoters have been studied, particularly in the ubiquitous bacterial host E. coli. In von Gabain, et al., PNAS (USA), 76, pages 189-193 (1979), several E. coli. phage and plasmid DNAs were characterized by the efficiency of the RNA polymerase binding site. The study compared various fragments of the following DNA species: bacterophage T5 fragments A through H, J, K, and M; bacteriophage lambda fragments A-F; bateriophage fd, fragments A and B; bacteriophage T7 intact complete fragment; plasmid pSC101, HINDIII and EcoRI fragments; and plasmid pML21, the entire fragment and fragments A and B. The promoters from the early region of bacteriophage T5 were shown to be the strongest, based upon their ability to complex with or bind RNA polymerase. The T5 HindIII K and N fragments were shown to have rates of complex formation at least ten times greater than the other promoters analyzed in this study.
In other research, promoters from various bacterial and viral sources have been cloned in E. coli and their signal strength in vivo studied using expression from distal promoterless sequences encoding .beta.-galactosidase or other proteins as an indication of promoter activity [see, Casadaban, M. J., et al., J. Molecular Biology, 138, pages 179-207 (1980); and West, R. W., Jr., et al., Gene, 9, pages 175-193 (1980)]. It is considered likely, based on the above studies, that the T5 promoters, particularly those residing on HindIII restriction fragments K and N, are at least an order of magnitude stronger than any promoters currently in use in recombinant DNA expression systems in E. coli.
The DNA sequences for P25, P26, P28 and P207, promoters which reside on the HindIII N fragment of the T5 viral genome, have been published [see, Bujard, H., TIBS, 5, pages 274-278 (1980) and Bujard, H., et al., in Promoters: Structures and Function, (R. Rodriguez, M. Chamberlin, eds.) Praiger, N.Y., pp. 121-140 (1982)]. Gentz, et al. [Gentz, R., PNAS (USA), 78, 4936-4940 (1981)] describes a series of experiments designed to evaluate relative promoter and terminator sequence strengths wherein two marker genes are mounted on a plasmid with a terminator sequence in an intermediate position and wherein provision is made both for incorporation of alternative terminator sequences and for alternative promoter sequences. In one of the plasmid constructions effected, a 212 base pair sequence presumptively including an early T5 promoter (designated P207) was inserted upstream of a promoterless .beta.-galactosidase gene fragment provided with an fd coliphage transcription terminator sequence at a locus 3' to the protein coding region. Despite the presence of at least a residual portion of the normal lac operator sequence intermediate the promoter and the .beta.-galactosidase gene fragment, no mention of potential regulation of T5 promoter function by IPTG "induction" of the lac operator was made.
It is apparent that the T5 bacteriophage promoters could be of significant value in increasing the yield of exogenous proteins expressed in E. coli by recombinant DNA methods, provided that their strength can be harnessed through regulation by either operator sequences or transcription terminator sequences, or, preferably, both. In the absence of such regulation, it is expected that expressing genes under T5 promoter control would be difficult or would unnecessarily tax the metabolic abilities of the cells containing the T5 promoter control gene. Excessive and premature transcription and expression of a selected exogenous gene under T5 promoter control, for example, is likely to disable transformed cell metabolism so that the cells simply would not grow well in culture. Likewise, T5 promoter initiated transcription of DNA sequences beyond the desired protein coding region of a plasmid (e.g., transcribing through and past a structural gene in a circular plasmid) could be highly watseful of cellular energy resources and consequently counterproductive. To date, however, the provision of suitable regulation of T5 promoters in the microbial expression of exogenous polypeptides has not been described.
There thus continues to be a need in the recombinant DNA arts for fully operative methods and materials for enhancing microbial expression of exogenous polypeptides through use of strong promoters such as those extant in the T5 bacteriophage genome in a context permitting appropriate regulation.